Evaluation of the intervertebral neck injury criterion using simulated rear impacts

The Intervertebral Neck Injury Criterion (IV-NIC) is based on the hypothesis that intervertebral motion beyond the physiological limit may injure spinal soft tissues during whiplash, while the Neck Injury Criterion (NIC) hypothesizes that sudden changes in spinal fluid pressure may cause neural injury. Goals of the present study, using a biofidelic whole cervical spine model with muscle force replication, were to correlate IV-NIC with soft-tissue injury, determine the IV-NIC injury threshold, and compare IV-NIC and NIC. Using a bench-top apparatus, rear-impacts were simulated at 3.5, 5, 6.5, and 8 g horizontal accelerations of the T1 vertebra. Pre- and post-whiplash flexibility tests measured the soft tissue injury threshold, i.e. significant increases in the intervertebral neutral zone (NZ) or range of motion (ROM) above corresponding baseline values. Extension IV-NIC peaks correlated well with NZ and ROM increases at C0-C1 and at C3-C4 through C7-T1 (r=0.64 and 0.62 respectively, p<0.001). Average IV-NIC injury thresholds (95% confidence limits) varied among the intervertebral levels and ranged between 1.5 (1.1, 1.9) at C5-C6 and 3.4 (2.4, 4.4) at C7-T1. The NIC injury threshold was 8.7 (7.7, 9.7) m2/s2, substantially less than the proposed threshold of 15 m2/s2. Results support the use of IV-NIC for determining the cervical spine injury threshold and injury severity. Advantages of IV-NIC include the ability to predict the intervertebral level, mode, severity, and time of the cervical spine soft-tissue injury.     

Prophylactic effects of an N- and L-type Ca2+ antagonist, cilnidipine, against cardiac hypertrophy and dysfunction in stroke-prone, spontaneously hypertensive rats

To clarify the beneficial effects of cilnidipine, an L- and N-type calcium channel blocker, which were clinically observed against diastolic dysfunction in hypertrophied hearts of hypertensive patients, we investigated the effects of cilnidipine on cardiac remodeling and enhanced gene expression in stroke-prone, spontaneously hypertensive rats in comparison with that of captopril, a well-known angiotensin-converting enzyme inhibitor, at threshold doses with little blood pressure lowering effect. The expression of type III collagen and beta/alpha-myosin heavy chain as well as transforming growth factor-beta, and basic fibroblast growth factor were suppressed by both treatments, indicating the prevention or amelioration of cardiac dysfunction. Such beneficial effects were much more intense with cilnidipine treatment than in captopril. These results indicate that Ca2+ is a key factor in the pathogenesis of cardiac remodeling in hypertension. One possible beneficial effect of cilnidipine in the prevention of cardiac dysfunction may be due to the decreased amount of growth factors such as transforming growth factor-beta and basic fibroblast growth factor via direct action for Ca2+ influx and also via inhibition of local renin-angiotensin system in the myocardium.     

Functional characterization of the thi1 promoter region from Arabidopsis thaliana

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.     

Characterization of the organic cation transporter SLC22A16: a doxorubicin importer

Specific efflux transporters, such as P-glycoprotein, have been shown to confer drug resistance by decreasing the intracellular accumulation of anticancer drugs. Understanding influx transporters, as well as efflux transporters, is essential to overcome this resistance. We report the expression profile and pharmacological characterization of an organic cation transporter, SLC22A16. The results of our experiments indicate that SLC22A16 is a mediator of doxorubicin uptake in cancer cells. Quantitative real-time RT-PCR analyses show that SLC22A16 is expressed in primary samples taken from patients with acute leukemia. Xenopus oocytes injected with SLC22A16 cRNA import doxorubicin, a widely used anticancer drug for hematological malignancies, in a saturable and dose-dependent manner. The apparent Km value for doxorubicin import was 5.2+/-0.4 microM. In cytotoxic assays, stable transfectants of leukemic Jurkat cells overexpressing SLC22A16 cells became significantly more sensitive to doxorubicin (2 microM) treatment. Characterization of SLC22A16 will help in designing novel therapies targeting hematological malignancies.     

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.     

Genome-wide application of RNAi to the discovery of potential drug targets

Progress is being made in the development of RNA interference-based (RNAi-based) strategies for the control of gene expression. It has been demonstrated that small interfering RNAs (siRNAs) can silence the expression of target genes in a sequence-specific manner in mammalian cells. Various groups, including our own, have developed systems for vector-mediated specific RNAi. Vector-based siRNA- (or shRNA) expression libraries directed against the entire human genome and siRNA libraries based on chemically synthesized oligonucleotides now allow the rapid identification of functional genes and potential drug targets. Use of such libraries will enhance our understanding of numerous biological phenomena and contribute to the rational design of drugs against heritable, infectious and malignant diseases.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

Triggering of TLR3 by polyI:C in human corneal epithelial cells to induce inflammatory cytokines

Epithelial cells of the ocular surface are key in the first-line defense as a part of the mucosal immune system against pathogens. We investigated whether polyI:C induces the production by human corneal epithelial cells (HCEC) of pro-inflammatory cytokines and IFN-beta, and whether Toll-like receptor (TLR)-3 expression is amplified by polyI:C. TLR3 was expressed on the surface of HCEC. Stimulation with polyI:C elicited the elevated production and mRNA expression of IL-6 and IL-8 in HCEC. While polyI:C induced IFN-beta, far stronger than human fibroblasts, and TLR3 gene expression in HCEC, LPS stimulation did not. Similarly, polyI:C, but not LPS, induced the gene expression of IkappaBalpha and MAIL, members of the IkappaB family, in HCEC. The innate immune response of HCEC is distinct from that of immune-competent cells, and we suggest that this is indicative of the symbiotic relationship between corneal epithelium and microbes inhabiting the ocular surface.     

Measurement of the carbohydrate-binding specificity of lectins by a multiplexed bead-based flow cytometric assay

Carbohydrate binding underlies many cell recognition events. Here, we describe a multiplexed glyco-bead array method for determining the carbohydrate-binding specificities of plant lectins using a bead-based flow cytometric analysis. N-glycans including high mannose, hybrid, and complex types and O-glycans from glycoproteins were immobilized on multiplexed beads, and the specificities of 13 kinds of sugar chains were monitored within 2 h in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples and allows the reliable and simultaneous elucidation of sugar-binding properties under identical conditions.